Ethyl pyruvate (EP) is the ester formed from pyruvic acid and ethanol. In preliminary studies, we have documented that EP ameliorates intestinal and hepatic injury or improves survival when it is used as a therapeutic agent to treat rodents subjected to mesenteric ischemia and reperfusion, hemorrhagic shock, endotoxemia, or polymicrobial bacterial sepsis. In addition, we have demonstrated that EP is an effective scavenger of reactive oxygen species (ROS), and we have shown that this compound is also an anti-inflammatory agent that inhibits activation of the pro-inflammatory signaling factors, NF-KappaB and p38 mitogen-activated protein kinase. EP also inhibits release of a novel cytokine, high mobility group box 1 (HMGB1). Prompted by these exciting observations, we propose to carry out a series of experiments that are designed to better elucidate the mechanism(s) responsible for the anti-inflammatory and therapeutic effects of EP. The work will be organized under three Specific Aims. Aim 1 is to test three broad hypotheses that might account for the beneficial effects of EP. These hypotheses are: the ROS Hypothesis, the Alkylation Hypothesis, and the Glutathione (GSH) Depletion Hypothesis. The ROS Hypothesis proposes that EP's ability to function as an antioxidant accounts for its cytoprotective and anti-inflammatory effects. The Alkylation Hypothesis proposes that EP functions as an electrophile that alkylates key thiol groups and thereby inactivates important signaling or effector molecules, such as subunits of the transcription factor, NF-KappaB, or various caspases involved in the process of apoptosis. The GSH Depletion Hypothesis, a variant of the Alkylation Hypothesis, proposes that EP alkylates GSH. Depletion of GSH shifts the cellular redox balance in a way that favors the formation of mixed disulfides with NF-KappaB subunits and thereby interferes with signaling via this pathway. Aim 2 is to carry out more detailed studies on the effects of EP on NF-KappaB activation. Aim 3 is to investigate the mechanisms responsible for inhibition of HMGB 1 release by EP. All of these aims will be carried out using a combination of molecular and pharmacological approaches and the studies will employ both in vitro (cell culture) and in vivo (animal model) approaches. Achieving a better understanding of the mechanisms responsible for the anti-inflammatory and therapeutic effects of EP may permit identification of novel cellular pathways involved in the innate immune response.